Hyaluronan (HA) is well known as a constituent of connective tissue extracellular matrices. Recent studies have also shown its abundance in stratified squamous epithelia, including the epidermis of skin. HA is often anchored to CD44, a ubiquitous, abundant, and structurally/functionally important surface receptor that displays HA binding site(s). Our preliminary findings indicate that both HA and CD44 participate in Ca 2+ mobilization and keratinocyte differentiation. Further analyses show that Rho/Rac and Src kinase signaling are closely coupled with HA/CD44-induced Ca 2+ mobilization, cytoskeletal function and keratinocyte differentiation. In addition, our data reveal that several keratinocyte differentiation markers (e.g. involucrin and filaggrin) are significantly reduced in the skin of CD44-knock out (k/o) mice as compared to CD44-wild type mice. These observations suggest the importance of CD44 in normal epidermal physiology and keratinocyte differentiation. In this proposed project we plan to test the hypothesis that CD44 plays an important role in regulating Ca 2+signaling and differentiation. Specifically, CD44 interaction with Rho-activated Rho-Kinase (ROK), Rac1-activated PLCT1 and Src kinases promotes IP3 receptor-mediated Ca2+ mobilization and cortactin-mediated cytosketetal reorganization/assembly leading to keratinocyte differentiation. Specifically, we will use a variety of biochemical, molecular biological techniques and immunohistochemical staining to elucidate HA-CD44 interaction with various signaling molecules [e.g. Rho-Kinase (ROK), PLCT1 and Src kinases] in regulating Ca 2+ signaling, and cytoskeleton reorganization/assembly and keratinocyte differentiation. Special emphasis will be placed on HA-mediated CD44-RhoA signaling and ROK in IP3 receptor-mediated Ca2+ mobilization during keratinocyte differentiation. We will also investigate HA-mediated CD44-Rac1 signaling and PLCgamma1 activation in regulating IP3 production, Ca 2+ mobilization and keratinocyte differentiation. In addition, we will analyze HA/CD44 interaction with c-Src/fyn kinases in regulating cortactin-actin binding and keratinocyte differentiation. Finally, we will evaluate the functional ramifications of CD44 interactions with various signaling molecules (e.g. ROK, PLCgamma1 and Src kinases) with respect to Ca 2+ signaling and cytoskeletal function and keratinocyte differentiation using cultured keratinocytes and/or skin tissues isolated from CD44-wild type and knock-out (k/o) mice exposed to HA. We believe that the new information obtained from this proposal may establish an important role for HA/CD44-mediated cellular signaling (e.g. Rho/Src & Ca 2+signaling) and cytoskeletal activation during keratinocyte function and normal epidermal development. A variety of skin diseases including psoriasis, squamous cell carcinoma (SCC) or basal cell carcinoma (BCC) have been shown to involve abnormal/defective HA-CD44 interactions in epidermal keratinocytes. Thus, dissection of HA and CD44-mediated intracellular events (e.g. Rho/Src activation, Ca 2+ signaling and cytoskeleton-regulated keratinocyte differentiation) should aid significantly our understanding of the keratinocyte biology underlying the clinical behaviors of HA/CD44-related skin diseases.